Project Summary The long-term objective of this project is to develop a new class of broad spectrum antibiotics, focused on Gram-negative bacteria. Inspired by a natural product, this class targets an unexploited binding site of the bacterial ribosome. With ever increasing reports of resistance to frontline therapies addressing Gram- negatives, there is a critical need for new therapies, yet very little can be found in the development pipeline. As a result, any new therapeutic that targets Gram-negative bacteria will address an unmet medical need. A viable approach to discover new therapeutic leads is re-evaluation of existing, but under-scrutinized classes of natural products. Through this approach a natural product scaffold was identified as a lead for a program directed toward antibiotics for tuberculosis. Initial evaluation of activity against other bacteria indicated that the antibacterial spectrum was limited to Mycobacterium spp. However, based on the chemical structure and related compounds, we expected to be able to generate analogs with more broad spectrum activity. In collaboration with Tom Steitz's lab at Yale, the structure of the initial scaffold and two analogues bound to the ribosome were recently solved. The structural studies revealed that the natural product lead and analogues bind to a highly conserved region of the peptidyl transferase center (PTC) in a manner that appears to convey prokaryotic selectivity and which has not been exploited in current therapeutics. Targeting this highly conserved region is expected to lead to slow rates of resistance. This structural information was used to generate two more potent analogues with favorable physicochemical properties. In a very preliminary SAR campaign of <30 compounds, we re-engineered a portion of our lead scaffold for both synthetic simplicity and stability to provide two analogs that introduce activity beyond Mtb, to both MRSA and E. coli, and do not exhibit cytotoxicity to eukaryotic cells (IC50 >100 M). Since the original submission, analogs active against K. pneumoniae, including a drug-resistant strain, have been produced. This two-year Phase I project will demonstrate feasibility for developing this lead scaffold as an effective broad spectrum antibiotic using the following aims. Aim 1 will develop new compounds to optimize potency and further refine the SAR using the co-crystal structures to guide medicinal chemistry efforts. Aim 2 will define biochemical and microbiological activity of analogs by evaluating activity against prokaryotic and eukaryotic protein synthesis, and a panel of bacteria representing Gram-negative and positive organisms, including antibiotic-resistant strains. Preliminary ADME-Tox will be evaluated in vitro in Aim 3. Phase I will culminate in Aim 4 determining the MTD to guide dosing that will be used to demonstrate proof-of-concept reduction in bacterial burden in a mouse thigh infection model.